Five million patients suffer from heart failure in the United States. Clinical trials of stem cell therapy, initiated because of the severity of this problem, have yielded modest results. Understanding the epigenetic programs that induce differentiation of a naive somatic adult-derived stem cell into a cardiomyocyte is an important step in developing stem cell therapy. Our application tests components of this epigenetic process in human mesenchymal stem cells (hMSCells), because of their potential use in clinical trials and the ability to generate them from the patient. The proposed studies are based on our ongoing successful collaboration between investigators at Duke University, University of North Carolina at Chapel Hill, and East Carolina University. This collaboration has led to our General Hypothesis: Nuclear Ca2+ oscillations induce hMSCells to differentiate into cardiomyocytes. More specifically, shared cytosolic conduits, gap junctions, between adult-derived stem cells and adjacent contracting cardiomyocytes provide the route by which a signal from the cardiomyocyte enters the stem cell. This signal is translated into de novo nuclear Ca2+ oscillations in the stem cell and activation of a cardiac gene program. We propose that the following sequential events drive naive stem cell differentiation into a cardiomyocyte: shared functional connexin 43 (Cx43)-derived gap junctions develop between the stem cell and an adjacent contracting cardiomyocyte (Cx43 is the dominant isoform in cardiomyocytes and hMSCells);de novo Ca2+ oscillations develop in the stem cell cytoplasm ([Ca2+]c) and nucleus ([Ca2+]n) that are synchronous with the cardiomyocyte cytosolic calcium ([Ca2+]i) transients. The [Ca2+]n oscillations are mediated by inositol trisphosphate receptor 1 (the hMSCell nuclear envelope IP3R is IP3R1);the expression of Ca2+- signaling dependent effectors, CaMKIV and the newly described regulator of cardiac gene expression, calmodulin binding transcription factor (CAMTA1), and cardiac genes become up-regulated and the stem cell acquires a cardiomyocyte phenotype. We will use in vitro and in vivo studies to test our hypothesis. PUBLIC HEALTH RELEVANCE: In the proposal, we attempt to understand the epigenetic basis underlying the process that induces a naive somatic adult-derived stem cell to differentiate into a cardiomyocyte. Our general hypothesis is nuclear Ca2+ oscillations induce hMSCells to differentiate into cardiomyocytes. We study this in co-cultures with neonatal cardiomyocytes in vitro and in vivo in the mouse heart.